Developing technologies to address fundamental questions about second messenger signaling Summary Microbiology has witnessed a renaissance in the field of nucleotide-derived second messenger signaling during the last decade. These intracellular signal molecules allow bacteria to sense and adapt to changing environmental conditions. The second messengers cyclic adenosine monophosphate (cAMP) and guanosine penta/tetra phosphate (p/ppGpp) have long been studied for their roles in gene regulation, catabolite repression, and the stringent response. However, the appreciation for another second messenger, cyclic diguanosine monophosphate (c-di-GMP) and its role in motility and biofilm formation has recently exploded. Moreover, three new bacterial second messengers (cyclic diadenosine monophosphate (c-di-AMP), cyclic guanosine monophosphate (cGMP), and now cyclic GMP-AMP (cGAMP)) have been recently discovered in bacteria. It is clear that cAMP and p/ppGpp were merely the tip of the proverbial second messenger iceberg. Growing evidence in eukaryotes suggests that the pyrimidine ribonucleotide derivatives, cyclic cytosine monophosphate (cCMP) and cyclic uridine monophosphate (cUMP), can be detected and have biological functions, but these signals have not been detected or studied in bacteria. With this recent explosion of interest in second messengers, there remains fundamental questions to be addressed, and this proposal will test two novel hypotheses about second messenger signaling in bacteria. First, we hypothesize that bacteria utilize pyrimidine-derived second messengers. My laboratory has developed rapid, sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) methods to quantify c-di-GMP, c-di-AMP, and pGpG (the breakdown product of c-di-GMP) in over twenty bacterial species using the world-class mass spectrometry core facility at Michigan State University. In this grant, we propose to develop LC-MS/MS protocols to quantify all known nucleotide-derived second messengers and the novel pyrimidine second messengers cCMP, cUMP, and c-di-UMP. We will then extract nucleotides from different bacterial species across the bacterial phylogenetic tree and measure the presence and concentration of these nucleotide-based second messengers. Our second hypothesis is that c-di-GMP displays phenotypic heterogeneity in bacterial populations. This hypothesis will be tested by developing species- specific RNA biosensors to quantify c-di-GMP in diverse bacteria at the single-cell level. This proposal will test fundamental hypotheses about bacterial second messenger signaling and develop new LC-MS/MS and single- cell reporter technologies that will catalyze new areas of bacterial second messenger research.